T cell anergy has important implications in autoimmunity, transplantation and tumor immunity. Recently, we identified a novel TCR signaling defect in antigen-specific anergic T cells, namely, impaired palmitoylation and, consequently, lipid raft localization and function, of linker for activation of T cells (LAT). Proper localization and function of other T cell signaling proteins is also critically dependent on their palmitoylation. Although protein palmitoylation has been known for >30 years, its mechanistic basis and regulation has, until very recently, been poorly understood. The recent discovery of a novel, large (23 member) family of mammalian protein palmitoyl acyl transferases (PATs) represents a major breakthrough in this area. This advance and our more recent preliminary evidence that defective LAT palmitoylation may actually cause anergy serve as a driving force for this project. We propose to conduct exploratory/developmental studies in order to characterize the expression of PATs in T lymphocytes, identify LAT-reactive PAT(s), and analyze their potential role in T cell responsiveness and anergy. In Aim 1, we will use quantitative real-time PCR to determine the mRNA expression profile of 23 known mouse PATs in resting, activated and anergic T cells, and confirm expression of T cell-expressed PAT mRNAs at the protein level. In Aim 2, we will use expression vectors of all T cell-expressed PATs, and employ an established ectopic expression system in order to screen the ability of these PATs to enhance the palmitoylation of LAT. Identified LAT-reactive PATs will be further tested for their ability to palmitoylate a limited set of other palmitoylation substrates as a test of their relative substrate specificity. In Aim 3, we will select the best candidates PATs identified in previous aims for further functional analysis. Specifically, we will use a novel, highly efficient method for siRNA delivery in order to knock-down the expression of selected PATs, and analyze the effects of PAT silencing on the functional status of T cells, with emphasis on determining whether silencing of LAT-reactive PATs will induce a functional state resembling T cell anergy. Understanding the mechanism that underlies defective LAT palmitoylation and its association with T cell anergy may provide critical clues on how reversible protein palmitoylation regulates key aspects of T cell fate and function, and potentially implicate substrate-selective PATs as future drug targets in autoimmunity and other immunological diseases. PUBLIC HEALTH RELEVANCE: Anergy is an important form of immune tolerance, in which T lymphocytes of the immune system, despite being able to recognize an antigen, cannot generate an effective immune response to it. Strategies aimed at inducing or preventing T cell anergy have significant implications in autoimmunity, transplantation, and tumor immunity. Here we will investigate a novel pathway for anergy induction discovered by us, which consists of defective processing and function of a key molecule, LAT, which is essential for the development and proper function of T lymphocytes.